Time course of renal glutamate dehydrogenase induction during NH4Cl loading in rats

1992 ◽  
Vol 262 (6) ◽  
pp. F999-F1006 ◽  
Author(s):  
P. A. Wright ◽  
R. K. Packer ◽  
A. Garcia-Perez ◽  
M. A. Knepper
Keyword(s):  
Glutamate Dehydrogenase ◽  
Time Course ◽  
Renal Cortex ◽  
Ammonium Excretion ◽  
Mrna Levels ◽  
Outer Medulla ◽  
Acid Intake ◽  
Blood Ph ◽  
Outer Stripe ◽  
Acid Loading

To study mechanisms involved in renal glutamate dehydrogenase (GDH) regulation in response to systemic acid loading, we have measured blood pH, ammonium excretion, renal GDH mRNA levels, and GDH activity in rats. Acid intake (0.28 M NH4Cl in drinking water for 3 days) increased GDH mRNA levels in the renal cortex, but had no effect in the outer stripe of the outer medulla, inner stripe of the outer medulla, or the inner medulla. Rats were subjected to a step change in acid intake by alkali loading for 3 days (7.2 meq NaHCO3 per day in food slurry) and shifting to acid loading for up to 7 days (7.2 meq NH4Cl in food slurry). Ammonium excretion rose rapidly, increasing by 14-fold in the first 24-h period and 38-fold in the second 24-h period. Cortical GDH mRNA levels were increased relative to alkali-loaded values by 3.7-fold in 24 h, 4.3-fold in 4 days, but only 2.2-fold in 7 days. GDH activity was unchanged after 24 h of acid intake, but was significantly increased after 48 h. We concluded the following: 1) GDH mRNA is present in all regions of the kidney, but levels increase in response to acid loading only in the renal cortex; 2) GDH mRNA levels increase within 1 day after the initiation of acid loading, but the associated increase in functional enzyme activity takes 2 or more days; and 3) the large increases in ammonium excretion that occur in the first day after initiation of acid loading are not dependent on increased GDH activity.

Temporal induction of clusterin in cisplatin nephrotoxicity.

1997 ◽  
Vol 8 (2) ◽  
pp. 302-305
Author(s):  
J R Silkensen ◽  
A Agarwal ◽  
K A Nath ◽  
J C Manivel ◽  
M E Rosenberg
Keyword(s):  
Serum Creatinine ◽  
Time Course ◽  
Tissue Injury ◽  
Kidney Tissue ◽  
Sprague Dawley ◽  
Outer Medulla ◽  
Creatinine Concentration ◽  
Cisplatin Nephrotoxicity ◽  
Tubular Necrosis ◽  
Outer Stripe

Clusterin is a ubiquitous glycoprotein induced in many organs, including the kidney, at times of tissue injury and/or remodeling. It is speculated in this study that clusterin preserves cell interactions that are otherwise perturbed by renal insults. The purpose of this study was to examine clusterin expression after cisplatin nephrotoxicity, a model characterized by a delayed time course of injury and a well-defined site of that injury (proximal tubule). Sprague-Dawley rats were treated with intravenous cisplatin (6 mg/kg) or vehicle. Serum creatinine concentrations were measured and kidneys harvested at 1, 2, and 5 days. Marked induction of clusterin mRNA was seen only at 5 days, a time when serum creatinine concentration was the highest. Histology of kidney tissue 5 days after cisplatin administration revealed marked tubular necrosis localized to the outer stripe of the outer medulla, a region rich in proximal tubules. Immunohistochemistry and in situ hybridization at 5 days demonstrated clusterin primarily in the inner stripe of the outer medulla. In conclusion, expression of clusterin follows renal injury with cisplatin at a time corresponding to the morphologic evidence of tubular necrosis and cell detachment; quite surprisingly, such expression occurs at a site distant from the primary injury.

Enhanced transcription of metallothionein genes in rat kidney: effect of uninephrectomy and compensatory renal growth

1995 ◽  
Vol 268 (4) ◽  
pp. F643-F650 ◽  
Author(s):  
R. K. Zalups ◽  
J. Fraser ◽  
J. Koropatnick
Keyword(s):  
Renal Cortex ◽  
Rat Kidney ◽  
Hepatic Tissue ◽  
Renal Growth ◽  
Outer Medulla ◽  
Compensatory Renal Growth ◽  
Intracellular Regulation ◽  
Outer Stripe ◽  
Remnant Kidney

Metallothioneins (MTs) have been implicated in the intracellular regulation of essential metals in eukaryotic cells, and increased expression of MT genes has been demonstrated during the growth and proliferation of cells. To explore the expression of MT in somatic cells undergoing growth (hypertrophy) in the kidney in situ, we measured the rates of transcription of the genes for MT-1 and MT-2, measured the levels of mRNA for MT-1 and MT-2, and measured the concentration of MT-1 and MT-2 protein in samples of renal (and hepatic) tissue from uninephrectomized (NPX) and sham-operated (SO) rats 15 days after surgery. The rates of transcription of the genes for MT-1 and MT-2 were found to be enhanced significantly in the remnant renal mass, particularly in the cortex and outer stripe of the outer medulla, and in the liver, after uninephrectomy and after 15 days allowing for compensatory renal growth. Increased accumulation of mRNA for MT-1 and MT-2 also occurred in the cortex and outer stripe of the outer medulla of the remnant kidney and in the liver in the NPX rats. Increased concentration of MT-1 and MT-2 protein (measured by radioimmunoassay), at the level of the whole kidney, renal cortex, and liver, was another feature detected in rats after uninephrectomy and 15 days of compensatory renal growth. These findings indicate that compensatory renal growth in response to uninephrectomy is associated with the induction of the expression of MT genes in the renal cortex and outer stripe of the outer medulla, as well as in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene expression of growth-related proteins and ECM constituents in response to unilateral nephrectomy

1992 ◽  
Vol 262 (3) ◽  
pp. F389-F396 ◽  
Author(s):  
T. Nakamura ◽  
I. Ebihara ◽  
Y. Tomino ◽  
H. Koide ◽  
K. Kikuchi ◽  
...  
Keyword(s):  
Time Course ◽  
Renal Cortex ◽  
Core Protein ◽  
Control Level ◽  
Unilateral Nephrectomy ◽  
Nuclear Antigen ◽  
Type Iii Collagen ◽  
Type I ◽  
Mrna Levels ◽  
Related Proteins

To identify the specific regulatory mechanism associated with the events following unilateral nephrectomy, we measured the levels of mRNA encoding for extracellular matrix (ECM) constituents, for protooncogenes, and for proliferating cell nuclear antigen (PCNA) in renal cortex and glomeruli. One hour after left nephrectomy, c-jun and c-fos mRNA levels in renal cortex increased rapidly and then decreased rapidly to the control level, whereas c-myc and PCNA mRNA levels showed a slower and more sustained increase, with a peak at 6 h after nephrectomy, and then decreased to the control level after 7 days. mRNA levels for basement membrane components including alpha 1-chain of type IV collagen, laminin B1 and B2 chains, and heparan sulfate proteoglycan core protein were significantly increased in renal cortex at 12 h after nephrectomy, whereas those for interstitial collagens including alpha 1-chains of type I and type III collagen were unchanged following nephrectomy. On the other hand, the glomerular expression of all genes examined in this study showed little change during the experimental period. These results suggest that the time course of mRNA expression of ECM constituents is different from that of growth-related proteins in renal cortex and that glomerular mRNA levels for these components may not be associated with renal hypertrophy in the early stages following unilateral nephrectomy.

Renal expression and urinary excretion of aquaporin-2 in postobstructive uropathy in rats

2021 ◽  
pp. 1-8
Author(s):  
Anabel Brandoni ◽  
Adriana M. Torres
Keyword(s):  
Time Course ◽  
Renal Cortex ◽  
Renal Medulla ◽  
Obstructive Nephropathy ◽  
Kidney Cortex ◽  
Mrna Levels ◽  
Whole Cell Lysate ◽  
Aquaporin 2 ◽  
Immunohistochemical Techniques ◽  
Apical Membranes

This work assessed the time course of water renal management together with aquaporin-2 (AQP2) kidney expression and urinary AQP2 levels (AQP2u) in obstructive nephropathy. Adult male Wistar rats were monitored after 1, 2, and 7 days of bilateral ureteral release (bilateral ureteral obstruction (BUO); BUO-1, BUO-2 and BUO-7). Renal water handling was evaluated using conventional clearance techniques. AQP2 levels were assessed by immunoblotting and immunohistochemical techniques. AQP2 expression in apical membranes was downregulated in BUO-1 rats and upregulated both in BUO-2 and BUO-7 animals. AQP2 protein expression in whole cell lysate fraction from kidney cortex and medulla were significantly decreased in all the experimental groups. Concomitantly, mRNA levels of AQP2 decreased in renal medulla of all groups and in renal cortex from BUO-1; however, in renal cortex from BUO-2 and BUO-7 a recovery and an increase in the level of AQP2 mRNA were, respectively, observed. BUO-7 group showed a significant increase in AQP2u. The alterations observed in apical membranes AQP2 expression could explain, at least in part, the evolution time of water kidney management in the postobstructive phase of BUO. Additionally, the AQP2u increase after 7 days of ureteral release may be postulated as a biomarker of improvement in the kidney function.

scholarly journals Expression of the B splice variant of NBCe1 (SLC4A4) in the mouse kidney

2018 ◽  
Vol 315 (3) ◽  
pp. F417-F428 ◽  
Author(s):  
Lijuan Fang ◽  
Hyun-Wook Lee ◽  
Chao Chen ◽  
Autumn N. Harris ◽  
Michael F. Romero ◽  
...  
Keyword(s):  
Splice Variant ◽  
Splice Variants ◽  
Immunoblot Analysis ◽  
Mouse Kidney ◽  
Wild Type ◽  
Rt Pcr ◽  
Outer Medulla ◽  
Bicarbonate Transporter ◽  
Outer Stripe ◽  
Acid Loading

Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study’s purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.

Endogenous EGF as a potential renotrophic factor in ischemia-induced acute renal failure

1993 ◽  
Vol 265 (3) ◽  
pp. F425-F434 ◽  
Author(s):  
R. P. Schaudies ◽  
D. Nonclercq ◽  
L. Nelson ◽  
G. Toubeau ◽  
J. Zanen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Time Course ◽  
Tissue Injury ◽  
Cell Turnover ◽  
Paraffin Sections ◽  
Outer Medulla ◽  
New Synthesis ◽  
Predominant Form ◽  
Outer Stripe ◽  
Epidermal Growth

The time course for the increases in soluble renal epidermal growth factor (EGF) after ischemia has been established. These elevated levels of EGF have been compared with the degree of tissue injury as well as the extent of cell proliferation in the recovering tissue. Levels of soluble immunoreactive EGF (irEGF) in control animals were 9.74 +/- 1.1 ng/g wet wt (n = 4-8 for all values) and rose to 83.9 +/- 30 ng/g within 12 h after injury. Soluble irEGF content peaked at 88.8 +/- 15 ng/g at 24 h postinjury and returned to control values by 72 h. We previously reported that trypsin digestion of crude renal membranes (CRM) generates rat EGF that is indistinguishable from that isolated from the submandibular gland. Initial levels of trypsin-releasable membrane-associated irEGF were 439 +/- 26 ng/g. These levels fell to 46.6 +/- 9.6 ng/g at 48 h after injury. The total renal EGF demonstrated an 80% decline 48 h after injury but returned to 50% of the initial values after 72 h representing significant new synthesis of EGF-containing proteins between 48 and 72 h postinjury. Immunohistochemical staining of kidney paraffin sections for EGF immunoreactivity demonstrated staining intensities that paralleled the amount of irEGF in the trypsin-digested CRM fraction, suggesting that the membrane-associated irEGF is the predominant form detected by this technique. Regenerative hyperplasia subsequent to tubular insult was monitored by immunostaining nuclei of S phase cells after pulse labeling with the thymidine analogue 5-bromo-2'-deoxyuridine. Cell proliferation was particularly prominent in the outer stripe of outer medulla of kidneys exposed to ischemia and reached a maximum (19-fold higher than the baseline value) 48 h after reperfusion. Renal cell turnover returned to control values by day 7. The observation that the peak in soluble EGF levels (24 h) precedes the peak in tubular regeneration (48 h) by 24 h is consistent with the hypothesis that EGF is one of the mitogenic signals triggering regenerative hyperplasia after renal injury.

scholarly journals Effect of starvation on glutamine ammoniagenesis and gluconeogenesis in isolated mouse kidney tubules

2002 ◽  
Vol 368 (1) ◽  
pp. 301-308 ◽  
Author(s):  
Agnès CONJARD ◽  
Virginie BRUN ◽  
Mireille MARTIN ◽  
Gabriel BAVEREL ◽  
Bernard FERRIER
Keyword(s):  
Amino Acid ◽  
Glutamate Dehydrogenase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Renal Cortex ◽  
Mouse Kidney ◽  
Glutamine Metabolism ◽  
Biological Research ◽  
Mrna Levels ◽  
Ammonium Ions ◽  
Stimulation Of

It has been shown recently that glutamine is taken up by the mouse kidney in vivo. However, knowledge about the fate of this amino acid and the regulation of its metabolism in the mouse kidney remains poor. Given the physiological and pathophysiological importance of renal glutamine metabolism and the increasing use of genetically modified mice in biological research, we have conducted a study to characterize glutamine metabolism in the mouse kidney. Proximal tubules isolated from fed and 48h-starved mice and then incubated with a physiological concentration of glutamine, removed this amino acid and produced ammonium ions at similar rates. In agreement with this observation, activities of the ammoniagenic enzymes, glutaminase and glutamate dehydrogenase, were not different in the renal cortex of fed and starved mice, but the glutamate dehydrogenase mRNA level was elevated 4.5-fold in the renal cortex from starved mice. In contrast, glucose production from glutamine was greatly stimulated whereas the glutamine carbon removed, that was presumably completely oxidized in tubules from fed mice, was virtually suppressed in tubules from starved animals. In accordance with the starvation-induced stimulation of glutamine gluconeogenesis, the activities and mRNA levels of glucose-6-phosphatase, and especially of phosphoenolpyruvate carboxykinase, but not of fructose-1,6-bisphosphatase, were increased in the renal cortex of starved mice. On the basis of our in vitro results, the elevated urinary excretion of ammonium ions observed in starved mice probably reflected an increased transport of these ions into the urine at the expense of those released into the renal veins rather than a stimulation of renal ammoniagenesis.

AQP3, p-AQP2, and AQP2 expression is reduced in polyuric rats with hypercalcemia: prevention by cAMP-PDE inhibitors

2002 ◽  
Vol 283 (6) ◽  
pp. F1313-F1325 ◽  
Author(s):  
Weidong Wang ◽  
Chunling Li ◽  
Tae-Hwan Kwon ◽  
Mark A. Knepper ◽  
Jørgen Frøkiær ◽  
...  
Keyword(s):  
Vitamin D ◽  
Collecting Duct ◽  
Urine Osmolality ◽  
Outer Medulla ◽  
Camp Phosphodiesterase ◽  
Aqp4 Expression ◽  
Pde Inhibitors ◽  
Aqp1 Expression ◽  
Outer Stripe ◽  
Inhibitor Treatment

The purpose of this study was to evaluate whether hypercalcemia is associated with downregulation of renal aquaporins (AQPs), including AQP1, AQP2, phosphorylated AQP2 (p-AQP2), AQP3, and AQP4, and if this is the case, to test whether cAMP-phosphodiesterase (PDE) inhibitor treatment can prevent AQP downregulation and prevent the development of polyuria. Vitamin D-induced hypercalcemia in rats was associated with increased urine output and reduced urine osmolality, consistent with previous findings (Levi M, Peterson L, and Berl T. Kidney Int 23: 489–497, 1983). Semiquantitative immunoblotting revealed a significant reduction in the abundance of inner medullary AQP2 (52 ± 6% of control levels), consistent with previous studies, and of AQP2, which is phosphorylated at the PKA phosphorylation consensus site serine 256 (p-AQP2; 36 ± 8%). Moreover, AQP3 abundance was also significantly decreased (45 ± 7 and 61 ± 6% of control levels in inner medulla and whole kidney, respectively). Consistent with this, immunohistochemistry demonstrated reduced AQP3 immunolabeling along the entire collecting duct. AQP4 expression was not reduced. Surprisingly, total kidney AQP1 abundance was also reduced (60 ± 6%). AQP1 expression was reduced in the cortex and outer stripe of the outer medulla (48 ± 7%; i.e., in proximal tubules). In contrast, AQP1 levels were not changed in the inner stripe of the outer medulla or in the inner medulla (i.e., descending thin limbs and vasa recta). Treatment with the cAMP-PDE inhibitors rolipram and milrinone in combination (inhibiting PDE IV and PDE III isoenzymes) at day 2 and onward completely prevented the hypercalcemia-induced downregulation of AQP2 and AQP3 (but not AQP1) and completely prevented the development of polyuria. In conclusion, AQP3, AQP2, and p-AQP2 are downregulated and are likely to play critical roles in the development of polyuria associated with vitamin D-induced hypercalcemia. Moreover, PDE inhibitor treatment significantly prevented the reduced expression of collecting duct AQPs and prevented the development of polyuria.

Abstract 315: Efficacy and Safety of Rivaroxaban in in vitro Experiments With the Endothelial Cells

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Kaname Seki ◽  
Yosuke Mizuno ◽  
Toku Sakashita ◽  
Jun Tanno ◽  
Shintaro Nakano ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Time Course ◽  
Umbilical Vein ◽  
Endothelial Damage ◽  
Coagulation Cascade ◽  
Elisa Assay ◽  
Mrna Levels ◽  
Factor X ◽  
Clotting Factors ◽  
Inflammatory Phase

Aim: Activated factor X (FXa) plays important roles in the thrombin generation and in inflammation, which is evoked during the endothelial damage. Although rivaroxaban is a selective FXa antagonist, it is one of the key therapies in ischemic heart disease, and yet its function in the state of inactivated coagulation cascade is uncertain. Rivaroxaban blocks FXa in the blood but not the tissue, while factor X is converted to FXa only when glutamic acid is changed to γ-carboxyglutamic acid by vitamin K following the intrinsic clotting factors and/or cellular injury activation. To uncover this aspect, we performed the following experiments. Methods and results: Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Co., Ltd. The cells were grown to 80% confluence and were treated with rivaroxaban (100nM, 500nM, 1000nM, 2000nM respectively) without FXa stimulation for 4 h, 10 h or 24 h. Cells and medium were collected and then their RNA was extracted from the cells. The qPCR of MCP-1, PAR1-4 and the DNA micro arrays (The GeneChip Human Gene 2.0 ST Array, Affymetrix) were performed. There was neither increased nor decreased gene expression significantly in either experimental time course of the qPCRs or the the DNA micro arrays. The ELISA assay of MCP-1 with medium showed non-activated MCP-1. As a next step, cells were treated with 100nM FXa and with/without rivaroxaban in same time course, and cells and medium were collected for further experiments. FXa evoked induction of mRNA levels for several pro-inflammatory cytokines including MCP-1 maximally at 4h, whereas MCP-1 was maximally evoked at 24 h in ELISA assay. Interestingly rivaroxaban inhibited both in all time course, at 4 hour inflammatory phase and at 24 hour inflammatory phase. Conclusion: Collectively, these results suggest that rivaroxaban may be safe in the inactivated coagulation state, and has the efficacy to attenuate the endothelial damage evoked by FXa and by pro-inflammatory cytokine genes.

Acid-Base Effects of Combined Renal Deletion of NBCe1-A and NBCe1-B

2022 ◽  
Author(s):  
Hyun-Wook Lee ◽  
Jill W. Verlander ◽  
Gary E Shull ◽  
Autumn N. Harris ◽  
I. David Weiner
Keyword(s):  
Proximal Tubule ◽  
Splice Variant ◽  
Molecular Mechanisms ◽  
Ammonia Excretion ◽  
Basal Diet ◽  
Acid Base ◽  
Outer Medulla ◽  
Ammonia Metabolism ◽  
Induced Changes ◽  
Acid Loading

The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deleting the A splice variant of the Na⁺-bicarbonate cotransporter, electrogenic, isoform 1 (NBCe1-A) partially blocks the effect of acidosis to increase urinary ammonia excretion, and this appears to involve the dysregulated expression of ammoniagenic enzymes in the proximal tubule (PT) in the cortex, but not in the outer medulla (OM). A second NBCe1 splice variant, NBCe1-B, is present throughout the PT, including the OM, where NBCe1-A is not present. The current studies determined the effects of combined renal deletion of NBCe1-A and NBCe1-B on systemic and proximal tubule ammonia metabolism. We generated NBCe1-A/B deletion using Cre-loxP techniques and used Cre-negative mice as controls. Since renal NBCe1-A and NBCe1-B expression is limited to the proximal tubule, Cre-positive mice had proximal tubule NBCe1-A/B deletion (PT-NBCe1-A/B KO). While on basal diet, PT-NBCe1-A/B KO mice had severe metabolic acidosis, yet urinary ammonia excretion was not changed significantly. PT-NBCe1-A/B KO decreased expression of phosphate-dependent glutaminase (PDG) and phospho­enol­pyruvate carboxy­kinase (PEPCK) and increased expression of glutamine synthetase (GS), an ammonia recycling enzyme, in PT in both the cortex and OM. Exogenous acid-loading increased ammonia excretion in control mice, but PT-NBCe1-A/B KO prevented any increase. PT-NBCe1-A/B KO significantly blunted acid loading-induced changes in PDG, PEPCK, and GS expression in the proximal tubule in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to proximal tubule ammonia metabolism in the OM and thereby to systemic acid-base regulation.

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